Plasmid construction and sgRNA design
Cas9 coding region of pX330 plasmid (Gifted from Dr. Feng Zhang, Addgene
accession no. 42230) was replaced with EGFP cDNA (Fig. 1B). EGFP
sequence was PCR amplified from pEGFP-N1 (Clontech cat# 6059-1) (Fig.
1C) using following primers: EGFP-F:
5’-GGCCACCGGT GATCCACCGGTCGCCACCAT-3’ (20bp) and EGFP-R: 5’-
GGCCGAATTC TTACTTGTACAGCTCGTCCATG-3’ (22bp) with AgeI site
at 5’-end and EcoRI site at 3’-end (AgeI and EcoRIare shown by underline). PCR was performed at 94°C for 4min, 24 cycles
of 94°C for 30s, 56°C for 30s, 72°C for 1min and 72°C for 10min. EGFP
PCR products were digested with AgeI and EcoRI (NEB) and
inserted into AgeI and EcoRI sites of pX330. Resultant
pX330-EGFP plasmid (Fig. 1B) was confirmed by sequencing. Oligos coding
for sgRNA targets were synthesized by Genewiz (Beijing, China), annealed
at 95°C for 5min and ramped down to 25°C (-5°C/min) and then subcloned
into BbsI sites of pX330-EGFP. BE4 plasmid (Fig. 1A) was gifted from
David Liu lab (Addgene access no. 100802). The sgRNAs were designed
using online platform https://benchling.com/ and all sgRNAs oligos
are listed in Supplementary Table 1.